Ticks and tick-borne pathogens in livestock from nomadic herds in the Somali Region, Ethiopia.

Between May 2006 and January 2007, blood samples and ticks were randomly collected from 220 nomadic animals from Filtu and Dollo Odo districts, Libaan zone, in the Somali Region of Ethiopia. Overall, 81.5% cattle, 98.2% camels, 53.4% goats and 61.1% sheep were infested by ixodid ticks. Collected ticks (n = 1,036) were identified as Rhipicephalus pulchellus (40.1%), R. pravus (25.8%), Amblyomma gemma (9.4%), Hyalomma rufipes (13.3%), H. truncatum (2.8%), H. impeltatum (1.2%) and H. dromedarii (0.5%); immature stages (6.1%) belonged to the genera Rhipicephalus and Amblyomma. Tick infestation burden was evaluated by the Tick Abundance Score method on 57 animals from Dollo Odo in August 2006, and it was significantly higher in cattle and camels than in small ruminants (p < 0.001). Reverse Line Blot Hybridisation was applied to detect Theileria, Babesia, Ehrlichia and Anaplasma spp. Five out of 50 blood samples from Filtu, four from cattle and, surprisingly, one from a camel, were positive for Theileria mutans and two from cattle for T. velifera. Adult ticks (n = 104) from both districts were tested and A. gemma from cattle were positive to T. velifera (1) and Ehrlichia ruminantium (5 samples). Positive E. ruminantium samples were also tested by PCR targeting pCS20 and 16S rRNA genes and submitted to DNA sequencing. The phylogenetic reconstruction of pCS20 fragment showed the presence of the Somali region sequences in the East-South African group. Our results are the first available on ticks and selected tick-borne diseases from the Somali region of Ethiopia and could be used as preliminary information for planning sustainable control strategies for tick and tick-borne pathogens in the study area and in neighbouring areas with similar socio-ecological features.

Detection of Rickettsia aeschlimannii and Rickettsia africae in ixodid ticks from Burkina Faso and Somali Region of Ethiopia by new real-time PCR assays.

In the framework of cooperation for development projects in Burkina Faso and Ethiopia, we collected ixodid ticks from cattle, small ruminants and camels. We optimized new TaqMan Probe real-time PCR assays to detect Rickettsia aeschlimannii and Rickettsia africae OmpA gene in the collected samples. Rickettsia africae was identified in 75.0% Amblyomma variegatum (95%CI: 56.6-88.5), while R. aeschlimannii in 24.0% Hyalomma truncatum (95%CI: 9.4-45.1) and 50.0% H. rufipes (95%CI: 29.9-70.0) collected from cattle in different provinces throughout Burkina Faso. Ticks from the Libaan zone, Somali Region of Ethiopia, were also infected by R. africae (28.5% prevalence in Amblyomma gemma, 95%CI: 14.7-46.0) and R. aeschlimannii (27.0% H. truncatum, 95%CI: 5.0-62.9; 88.3% H. rufipes, 95%CI: 60.5-99.3). All tested ticks were adults. The developed diagnostic tools were highly sensitive and enabled us to rapidly classify R. aeschlimannii and R. africae, which were identified in Burkina Faso and in the Somali Region of Ethiopia for the first time. Further studies are needed to assess the zoonotic risk and prevalence of infection in local human populations, who have high contact rates with ticks and their animal hosts.

Methylangelicins: new potential agents for the photochemotherapy of psoriasis. Structure-activity study on the dark and photochemical interactions with DNA.

The interactions both in the ground and in the excited state between various methylangelicins, previously prepared with the aim to increase the low photobiological activity of the parent angelicin 1, and DNA have been studied. In general, the new methylangelicins show an increased capacity to photobind monofunctionally to DNA and a parallel increment of photobiological activity in comparison with the parent 1. This increase appears to be connected with various factors, such as the augmented affinity toward DNA for the dark complex formation and the electronic effect connected with the introduction into 1 of one or two methyl groups. The new compounds, on the basis of their photobiological activity and their lack of skin phototoxicity, appear as possible agents for the photochemistry of skin diseases characterized by cell hyperproliferation.

4'-Methylangelicins: new potential agents for the photochemotherapy of psoriasis.

Three derivatives of angelicin (1) [4'-methyl-, 4,4'-dimethyl-, and 4',5-dimethylangelicin (2a-c)] have been prepared with the aim of obtaining new agents for the photochemotherapy of psoriasis. These compounds form a complex in the dark with DNA that shows an affinity for the macromolecule higher than that of the parent angelicin (1). A correlation between their octanol/water partition coefficients and the association constants of the complexes has been observed. Compounds 2a-c photobind to DNA to a much higher extent than 1 and also more effectively than 8-methoxypsoralen (8-MOP), taken as reference compound. When activated with UV-A, the three compounds strongly inactivate T2 phage and inhibit epidermal DNA synthesis in mice. Moreover, they show a mutagenic activity markedly lower than that of 8-methoxypsoralen on Escherichia coli wild-type strain. Due to its lack of skin phototoxicity, its low mutagenic activity, and its antiproliferative activity, 2c was chosen for clinical evaluation. It proved to be effective in clearing psoriasis in two patients.

6-Methylangelicins: a new series of potential photochemotherapeutic agents for the treatment of psoriasis.

The possible presence of methylpsoralens as undesired inquinants in synthetic methylangelicins has been avoided through a synthetic pathway starting from umbelliferones carrying a methyl group in the 6-position. The new 6-methylangelicins show a high affinity toward DNA, forming in the dark a molecular complex; the complexed angelicins under UV-A irradiation photobind effectively to the macromolecule, forming only monoadducts. The new compounds show an evident antiproliferative activity by inhibiting DNA synthesis on Ehrlich cells; great differences, however, can be seen between the various compounds. All the compounds are lacking of skin erythemogenic activity. Some of the new 6-methylangelicins, evaluated in terms of mutagenic activity, demonstrate to be less effective than 8-methoxypsoralen (8-MOP), used for a comparison. On the basis of antiproliferative activity, lack of skin phototoxicity, and low mutagenicity, two compounds have been chosen for clinical evaluation. The compounds tested on seven psoriatic patients by topical application and UV-A irradiation proved to be more effective than 8-MOP, used in the same conditions.

New monofunctional reagents for DNA as possible agents for the photochemotherapy of psoriasis: derivatives of 4,5'-dimethylangelicin.

With the aim of obtaining new agents for the photochemotherapy of psoriasis, we have prepared monofunctional reagents for DNA by starting from 4,5'-dimethylangelicin (2), an angular furocoumarin, and introducing in a 4'-(hydroxymethyl) (3), 4'-(methoxymethyl) (4), or 4'-(aminomethyl) group (5), in way analogous to what other authors have done previously on trioxsalen, a DNA bifunctional reagent. These new compounds form complexes with DNA in the ground state and by successive irradiation (UV-A) undergo monofunctional photoaddition to the macromolecule. Photobinding to DNA was highest for 3 and gradually lower for 4 and 5, respectively. These compounds do not form interstrand photocross-linkages in DNA and do not show any skin phototoxicity. Fluorimetric studies show that their 4',5' double bond is involved in the photoaddition to DNA. Their photobiological activity evaluated on Ehrlich ascites tumor cells and on T2 phages was strictly connected with their photobinding to DNA. The effect of the introduction of hydroxymethyl and methoxymethyl groups in angular 2 is somewhat similar to that previously described for trioxsalen: the introduction of an aminomethyl group in 2 markedly increases the affinity in the dark for DNA but under UV-A irradiation strongly inhibits photobinding to the macromolecule. By contrast, in the analogous derivative of trioxsalen both the affinity for DNA in the dark and the photobinding to DNA increased.

Angular furoquinolinones, psoralen analogs: novel antiproliferative agents for skin diseases. Synthesis, biological activity, mechanism of action, and computer-aided studies.

With the aim of obtaining new potential photochemotherapeutic agents, having increased antiproliferative activity and decreased undesired effects, we have prepared some new furoquinolinones. Two of them have been studied in detail: 1,4,6,8-tetramethyl-2H-furo[2,3-h]-quinolin-2-one (8), and 4,6,8,9-tetramethyl-2H-furo[2,3-h]quinolin-2-one (10). These compounds form a molecular complex with DNA, undergoing intercalation inside the duplex macromolecule, as shown by linear flow dichroism. The complexed ligands, by subsequent irradiation with UV-A light, photobind with the macromolecule forming only monocycloadducts with thymine with cis-syn configuration. In order to evaluate the electronic effects induced by the nitrogen atom in position 1 of 8, semiempirical calculations have been performed on both 4,6,4'-trimethylangelicin (TMA) and 8. The results obtained do not clearly differentiate between the two molecules which, at this level of approximation, show the possibility of photoreaction with both the 3,4- and 8,9-olefinic bonds for 8 and the 3,4- and 4',5'-bonds for TMA. In the lower energy conformation of intercalated 8, the furan ring is turned toward the minor groove of the polynucleotide, in such a way that photoreaction of this ring with thymine is favored. These compounds unexpectedly inhibit DNA and RNA synthesis in Ehrlich cells, in the dark. They also show a strong photoantiproliferative activity, 2 orders of magnitude higher than 8-methoxypsoralen (8-MOP), the most used drug for photochemotherapy. Their mutagenic activity on Escherichia coli is similar to that of TMA and 8-MOP. On the basis of these results, the compounds should deserve evaluation of their activity in the treatment of hyperproliferative skin diseases.

4',5'-substituted methylangelicins: photocycloadducts with pyrimidine bases of DNA.

The isolation and characterization of photocycloadducts with pyrimidine bases from DNA samples irradiated (365 nm) in the presence of four 4',5'-substituted methylangelicins was performed. All these furocoumarins yielded mainly the cis-syn furan-side cycloadduct with thymine. For 4',5'-dimethyl-, 5,4',5'-trimethyl- and 6,4',5'-trimethylangelicin this adduct was accompanied by two pyrone-side adducts (cis-syn and cis-anti), whereas the 4,4',5'-trimethyl derivative gave the furan-side adduct with cytosine. The characterization of the regio- and stereochemistry of the adducts was accomplished by 1H NOE (nuclear Overhauser effect) and 1H-13C HMBC (heteronuclear multiple-bond connectivity) spectroscopies. The formation of different cycloadducts in DNA by the various derivatives highlights the role of the methyl groups in determining the regio- and stereochemistry of the cycloaddition.

Photobiological properties of 1'-thieno-4,6,4'-trimethylangelicin.

Some photobiological properties of 1'-thieno-4,6,4'-trimethylangelicin (TTMA), a new isoster of 4,6,4'-trimethylangelicin (TMA) were studied in comparison with the parent compound. The TTMA absorbs UVA light and photobinds in vitro to DNA more efficiently than TMA; however, in Ehrlich cells in vivo TTMA linked to DNA to a lesser extent than the parent compound. In general, the formation of damage into DNA is in line with this last result: In fact, TTMA and TMA form equivalent amounts of interstrand cross-links (ISC) both in vitro in linearized PM2 DNA and in vivo in HeLa cells. In this system TTMA induces DNA-protein cross-links (DPC) more efficiently than TMA; on the contrary, no significant amounts of single-strand breaks were detected with both compounds. The antiproliferative activity of TTMA is consistent with these results, being only slightly more pronounced than that of TMA. Experiments carried out using double irradiation demonstrated that these drugs are capable of inducing antiproliferative effects by biphotonic reactions, including the formation of both ISC and DPC. Thus, replacement of the oxygen atom by a sulfur increases the UV absorption of the drug and its capacity to photobind to DNA in vitro but does not yield a comparable enhancement of its photosensitizing properties in vivo; this might be due to various reasons, for instance to an increase in the lipophilic character that could modify the behavior in vivo.

Receptor sites in DNA for the dark and photochemical interactions with 4,5'-dimethylangelicin, a potential agent for the photochemotherapy.

The study of the interactions in the ground state between 4,5'-dimethylangelicin, an angular furocoumarin, and various synthetic and natural DNA samples have evidenced the presence in the macromolecule of preferred sequences suitable for binding the small ligand. They are represented by an alternate sequence of purine and pyrimidine bases in each strand of the macromolecule, without difference between the two base pairs A-T and C-G. The study of the photochemical interactions between the same DNA samples and the 4,5'-dimethylangelicin shows that preferred sites are present in the macromolecule for the covalent addition of the furocoumarin to the macromolecule too. These sites however have more strict requirements than those useful for dark binding; they are in fact represented by alternate sequences of A-T in each strand such as those present in poly [d(A-T)] . poly-[d(A-T)]. Moreover fluorescence studies made on the same DNA samples irradiated in the presence of the furocoumarin suggest that the alternate C-G regions favour formation of 4'-5'-fluorescent adducts.

DNA damage and topoisomerase II inhibition induced by a benzopsoralen derivative.

The ability of 4-hydroxymethyl-4',5'-benzopsoralen (HMBP) to damage DNA of Chinese hamster ovary cells (CHO) and to inhibit the activity of topoisomerase II in vitro has been studied. This compound is characterized by a fourth ring condensed at the furan-side in the psoralen molecule. Contrary to other known furocoumarin derivatives, HMBP induces chromosomal aberrations in mammalian cells without UVA activation. The lesions induced in the dark by HMBP in DNA were studied by alkaline and neutral elution in CHO cells; comparable amounts of single-strand breaks and DNA-protein cross-links as well as the formation of double-strand breaks were detected. Moreover, HMBP appeared to inhibit the activity of mammalian topoisomerase II in vitro, in both the catenation and the decatenation assay. In these experiments the drug was effective only when it was pre-incubated with DNA substrate. These results are also consistent with the cytotoxic and mutagenic activity of HMBP in the dark, as tested on V79 Chinese hamster cells (V79/HGPRT system).

Pyrrolocoumarin derivatives: DNA-binding properties.

The spectroscopic and DNA-binding properties of a number of pyrrolocoumarin derivatives, including linear tricyclic, angular tricyclic, linear tetracyclic and angular tetracyclic compounds were investigated. The compounds we examined form non-covalent complexes with duplex DNA, probably of the intercalation type. The binding constants are comparable with the constants found for the furocoumarin analogues. Although for some of the compounds the photoreactivity with DNA is comparable with that of 8-MOP, pyrrolocoumarins behave as monofunctional reagents. This fact is explained in terms of an increased delocalization of the 4',5' double bond in the pyrrole moiety. Denaturation-renaturation experiments and HPLC analysis of the photoadducts confirm that pyrrolocoumarins are essentially monofunctional DNA-photobinding agents.

4,6,4'-Trimethylangelicin induces interstrand cross-links in mammalian cell DNA.

4,6,4'-Trimethylangelicin, a well-known effective photosensitizer described as a pure monofunctional reactant with DNA, can induce interstrand cross-links in mammalian cell DNA in vivo (about 15% relative to 8-methoxypsoralen), as observed using alkaline elution and Chinese hamster ovary cells. Experiments performed using the two-step irradiation method and HeLa cells support these data. In contrast with 4,6,4'-trimethylangelicin, 4'-methylangelicin and 4,4'-dimethylangelicin do not form interstrand cross-links. These results are consistent with those recently reported by Chen et al. (X. Chen, J. Kagan, F. Dall'Acqua, D. Averbeck and E. Bisagni, J. Photochem. Photobiol. B: Biol, 22 (1994) 51-57) using pBR322 and M13 DNA. The cross-linking ability of 4,6,4'-trimethylangelicin does not seem to be related to a particular feature of these DNAs but to the compound itself.

Photobiological properties of a new tetramethylfuroquinolinone.

1,4,6,8-tetramethyl-2H-furo[2,3-h]quinolin-2-one (FQ) is a new isoster of angelicin characterised by an extremely strong photosensitizing activity, which is several times higher than that of 8-MOP and 4,6,4'-trimethylangelicin (TMA). Following treatment with 1.2 microM FQ and a dose as low as 0.05 kJ m(-2) of UVA irradiation, survival (colony forming ability) of HeLa cells was abolished, while TMA and 8-MOP (even at five times the concentration for the latter) were practically ineffective. Upon UVA irradiation FQ induces various types of lesions in mammalian cells in DNA: single-strand breaks (SSBs), many monoadducts and covalent DNA-protein cross-links (DPC), but not interstrand cross-links (ISC). Using the two step irradiation procedure, DPC induced by FQ appeared to be severe lesions, having a high antiproliferative activity; their formation requires the successive absorption of two photons, thus, in this respect, resembling ISC formation. In spite of its higher capacity for damaging DNA, FQ showed a skin-phototoxicity potency very similar to 8-MOP. As some benzopsoralens, FQ induced a certain antiproliferative activity also in the dark, which was accompanied by the formation of double-strand breaks into DNA associated with DPC. This lesion is generally induced by topoisomerase inhibitors. On the basis of these features, FQ can be expected to show useful activities in photochemotherapy and photopheresis. However, before medical use careful studies on its genotoxicity are required.

Thienocoumarin derivatives: interaction with nucleic acids and synthetic polydeoxyribonucleotides.

This paper reports the photobiological properties of two new thienocoumarins, 4,6,9-trimethyl-2H-thieno[3,2-g]-1-benzopyran-2-one (compound I) and the 6,9-dimethyl-4-methoxymethyl-2H-thieno[3,2-g]-1-benzopyran-2-one (compound II). Cell growth inhibition studies have revealed significant antiproliferative potency on human tumor cell lines. The photoaddition process of these tritium-labeled derivatives was investigated using various nucleic acid structures (calf thymus DNA, bacterial DNA, and synthetic polydeoxyribonucleotides). The results obtained show that both compounds photobind to DNA to a higher extent than 8-MOP, taken as the reference drug. The capacity to form interstrand crosslinks into DNA helix was also evaluated. Interestingly, notwithstanding the lack of cutaneous phototoxicity, II revealed a good ability to induce diadduct formation.

2,6-Dimethyl-9-methoxy-4H-pyrrolo[3,2,1-ij]quinolin-4-one, a new compound with unusual photosensitizing properties.

Some photobiological properties of 2,6-dimethyl-9-methoxy-4H-pyrrolo[3,2,1-ij]quinolin-4-one (PQ) have been studied in comparison with 8-methoxypsoralen (8-MOP). In Ehrlich cells, PQ induced a moderate inhibition in DNA and RNA syntheses in the dark, which appeared to be more pronounced upon UVA irradiation. In contrast to 8-MOP, in the presence of UVA, PQ also affected protein synthesis. Likewise marked antiproliferative effects were also observed in the study of the clonal growth of CHO cells cultivated in vitro. Using alkaline elution and CHO cells, a moderate formation of single-strand breaks (SSBs) and of DNA-protein cross-links (DPCs) was observed by incubation in the dark; upon UVA irradiation the amount of both lesions increased greatly, whereas no inter-strand cross-links (ISCs) were formed. As expected, 8-MOP did not damage DNA in the dark, but induced SSBs, ISCs and DPCs in the presence of UVA. The induction of SSBs by both compounds seems to be directly related to a photochemical event rather than to incisions during DNA repair. As the induction of ISCs, and also the formation of DPCs by 8-MOP and UVA, appears to be based on a two-step reaction involving photo-bound 8-MOP-DNA moieties. In contrast, the formation of DPCs by PQ and UVA seems to involve photosensitization by free PQ molecules connected with SSB and DPC formation rather than with a DNA photo-binding activity. The PQ activity observed in the dark could probably be ascribed to a moderate inhibition of topoisomerases.

Synthesis and photobiological properties of acetylpsoralen derivatives.

In an investigation to find monofunctional reactants for DNA which can act as new agents in the photochemotherapy of psoriasis, we have synthesized and studied some methylpsoralen derivatives which contain an acetyl group at one of the two reactive sites of the furocoumarin skeleton (at the 3 or 5' positions). The compounds do not react easily with DNA; their photobiological properties (e.g. the lack of an ability to inhibit DNA synthesis in Ehrlich ascites tumour cells, to induce T2 phage sensitization and to induce erythema in guinea-pig skin) are exactly in line with this behaviour. Some interesting features are shown by 4,8-dimethyl-5'-acetylpsoralen: it is capable of producing a very large amount of singlet oxygen--an order of magnitude higher than the other compounds and 8-methoxypsoralen (used as reference). In spite of this property, 4,8-dimethyl-5'-acetylpsoralen is non-phototoxic to the skin, and its other photobiological properties appear to be in line with its lack of interaction with DNA rather than its enhanced singlet oxygen production.

4-Hydroxymethyltetrahydro- and 4-hydroxymethyl-benzopsoralen: synthesis and biological activity.

The synthesis and some biological properties of 4-hydroxymethyltetrahydro- and 4-hydroxymethylbenzopsoralen are reported. The two compounds exhibited activity in the dark and by UVA irradiation. The tetrahydrobenzo derivative was more effective than the corresponding aromatic compound. Benzopsoralens were more cytotoxic in malignant (HL60 and HeLa) cell lines than in normal ones (NCTC 2544). Their toxicity decreased in confluent cultures of NCTC 2544 cells.

Photobiological activity of certain new methylazapsoralens.

The photobiological activity of a series of psoralen isosters carrying a nitrogen atom at 8 position, new potential drugs for the photochemotherapy of hyperproliferative skin diseases, have been studied; the more active derivatives appeared to be 5,4'-dimethyl-8-azapsoralen and 3,4,4'-trimethyl-8-azapsoralen which induced a strong inhibition of DNA synthesis in Ehrlich ascites cells, very similar to that provoked by 8-methoxypsoralen, the furocoumarin at present used in photochemotherapy. Such compounds induced a small amount of inter-strand DNA cross-links and were non phototoxic when assayed on guinea-pig skin; however, both derivatives appeared to be highly mutagenic in E. coli WP2 TM6. This strain contains the plasmid R46 and it is proficient in DNA repair, and therefore monoadducts do not should be mutagenic in such a strain. Because the first steps of excision, which remove monoadducts, and of the main cross-link repair use the same enzymes (produced by the uvrABC complex), in the presence of a great number of monofunctional lesions, it is possible that there are not sufficient enzyme molecules for removing cross-links according this pathway, which could be repaired by a second one, uvrABC independent and based on glycosilase activity, which works at reduced levels and is much less accurate.

Synthesis and antiproliferative activity of furocoumarin isosters.

Furocoumarins are a group of natural and synthetic compounds, some of which are used for the photochemotherapeutic treatment of certain skin diseases. With the aim of decreasing the side-effects of furocoumarin photochemotherapy and possibly increasing the therapeutic effects of these drugs, some new furocoumarin isosters were synthesized. The chemical synthesis of furocoumarin isosters at the furan ring, such as pyrrolo-, thieno-, oxazolo- and triazolocoumarins are reported. For all these compounds the key intermediate is a properly functionalized coumarin, to which the third heterocyclic ring is condensed by successive steps. Linear and angular pyrrolo-, tetrahydrobenzo- and benzopyrrolocoumarins show reduced photobiological activity, but have a strong antiproliferative effect in the dark, probably through an interaction with topoisomerases. Oxazolocoumarins are too unstable to be studied; triazolocoumarins show very poor activity. Tienocoumarins have not yet been studied. Furocoumarin isosters at the benzene ring are represented by 8-azapsoralens. Chemical synthesis involves the key 8-azacoumarin in the place of coumarin. These compounds have photochemical and photobiological properties which are very similar to those of psoralens. In particular, 4,4',5'- is effective in the photochemotherapeutic treatment of psoriasis. Furoquinolinones and 4-azapsoralens are reported among the isosters at the pyrone ring. While the synthesis of pyrroloquinolinones involves properly functionalized 7-aminoquinolinones as key intermediate, that of 4-azapsoralen requires a quite different synthetic pathway, involving a properly functionalized benzofuran derivative as key intermediate. Furoquinolinones have dramatically high activity both in the dark and under light activation; 4-azapsoralens have not yet been investigated.